Narcolepsy, a Rapid Eye Movement (REM) sleep-related disorder, is characterized by excessive daytime sleepiness (EDS), cataplexy (a sudden loss of muscle tone triggered by emotional stimulation), and a cluster of other symptoms. Narcolepsy afflicts approximately 1 in 1000 Americans, and current treatments are symptomatic: cataplexy has classically been treated with anticholinergics, which have such undesirable side- effects that patients elect to live with cataplexy;and the debilitating sleepiness has been treated with amphetamine and other stimulant medications, which have abuse potential. More recently, modafinil has been used as a wakefulness-promoting therapeutic, and gamma-hydroxybutyrate, a controlled substance, has been approved to treat both cataplexy and EDS symptoms. Recently narcolepsy been shown to be a neurodegenerative disorder in which the hypocretin (orexin)-containing neurons of the hypothalamus specifically degenerate. The hypocretin peptides (Hcrt1 and Hcrt2) differentially bind two G protein-coupled receptors known as Hcrt receptor 1 and 2 (HcrtR1 and HcrtR2). The onset of narcolepsy symptoms is associated with a decline in Hcrt1 levels in cerebrospinal fluid, thought to be indicative of the ongoing loss of Hcrt-containing neurons in the PLH. Thus, although Hcrt replacement is a potential therapeutic regimen, no small molecule brain-penetrable HcrtR agonists currently exist. By screening a compound library synthesized at SRI International for potential HcrtR agonists, we have identified two hits, SRI-2757 and SRI-5653, that can serve as the basis for the iterative process of optimization using rational drug design and molecular modeling followed by drug synthesis and biological testing. Aim 1 will establish a structure-activity relationship for SRI-2757 and SRI-5653 and develop ligand-derived pharmacophore models and pharmacophore-based in silico screening of small-molecule libraries to identify other HcrtR agonists with novel scaffolds. Aim 2 will determine compound activity in vitro using FLIPR-based HcrtR functional assays and will evaluate the intestinal permeability and metabolic stability of selected analogs in models in vitro. Aim 3 will evaluate the efficacy of Hcrt agonists in vivo using behavioral assays in a mouse genetic model of narcolepsy. Since the Hcrt system is now recognized to be centrally involved in the maintenance of wakefulness, novel Hcrt agonists identified in this proposal may have broader indications than treating narcolepsy;for example, they may be used to prolong wakefulness in situations where sustained performance is necessary. Since Hcrt1 appears to have antinociceptive properties, Hcrt agonists may also be useful when analgesia is necessary, such as in acute injury, arthritis, and postoperative or neuropathic pain. In addition to these potential therapeutic roles, subtype-specific Hcrt agonists would be useful to help elucidate the biological functions of HcrtR1 and HcrtR2, since all reports to date have focused on the endogenous ligands, Hcrt1 and Hcrt2, and a few peptide analogs.